CN1165655A - Method of treating conditions with estrogen agonists - Google Patents

Abstract

Pharmaceutical compositions that comprise compounds of this formula are useful for treating or preventing Alzheimer's disease, premenstrual syndrome, perimenopausal syndrome, a deficiency of thrombomodulin, uterine fibrosis, excessive myeloproxidase activity, excessive thrombin, autoimmune disease, reperfusion damage of ischemic myocardium and insufficient testosterone.

Description

Method of treating disorders with estrogen agonists

Certain estrogen agonists have been reported to be very useful in inhibiting diseases associated with organ systems caused by estrogen agonists or antagonists. In particular, 2-phenyl-3-aroylbenzothiophenes and 1- (alkylaminoethoxyphenyl) -1-phenyl-2-phenylbut-1-enes, represented by raloxifene and tamoxifen, are widely used as estrogen agonists.

Raloxifene tamoxifen

Raloxifene is said to be effective in the treatment of: acne, U.S.5,439,923; alopecia, EP 0659414a 2; alzheimer's Disease, EP 0659418a 1; skin and vaginal atrophy, U.S.5,461,064; autoimmune diseases, EP 0664123; breast cancer, US4,418,068; breast disease, 0659419; cartilage degradation, US5,418,252; CNS (central nervous system) disorders (post-amenorrhea), 94 EP 0309470; endocrine target organ disease, US4,418,068; delayed puberty, US5,451,589; demyelinating diseases; US5,434,166; myelin pain disease; US5,434,166; dysmenorrhea, US5,446,053; endometriosis, US5,461,065; female infertility, EP 659429 a; fertility disorders; hirsutism, EP 0659414, a 2; hypoglycemia, EP635264 a 2; bulimia, US5,439,931; fertility inhibition, US5,462,949; LDL (low density lipoprotein) oxidation, EP 0664121 a; cholesterol-too-high, US5,464,845; lupus erythematosus, EP 0664125; impaired macrophage function, EP 659425 a 1; male infertility, EP 0659424a 1; myocardial infarction, ischemia, thromboembolic disorders, thrombin inhibition, EP 0664126; menopausal disorders, EP 0659415; menoxenia, US5,462,950; obesity, 94 EP 0309481; compulsive impulse disorders, EP 0659428; osteoporosis, US5,457,117; ovarian hypoplasia, US5,451,589; amenorrhea syndrome, US5,391,557; peripheral vasoconstriction, US5,470,883; post-amenorrhea CNS diseases, EP 0659415; premenstrual syndrome, US5,389,670; prostate cancer; hyperplasia of prostate; pulmonary hypertension, US5,447,941; reperfusion injury, journal of cardiology (j.am. cardiol)25, 189A (1993); resistant tumors, EP 0652004 a 1; restenosis, US5,462,937; rheumatoid arthritis, EP 0664125; seborrheic dermatitis, US5,439,923; loss of sexual function; precocious puberty, US5,451,590, thromboembolic expression, EP 0659427; turners syndrome, US5,441,966; uterine fibrosis, US5,457,116; and vasodilatory syndrome (post-amenorrhea), 94 EP 0309473.

Tamoxifen is widely used in the treatment of breast cancer and has been reported to be effective in treating the following diseases or conditions: hyperlipidemia, Drug theory (Drug Ther) 22/3, 109 (1992); ovarian cancer, journal of oncology (j. clin. oncol.), 11, No. 10, 1957-68, (1993); renal cell carcinoma, journal of radiology in uk (br.j. radio) 56, No. 670, 766-7 (1983); inhibition of the atherosclerotic factor homocysteine, env.j.cancer 29 binder 6, S110 (1993); metastatic melanoma, journal of clinical oncology 12, No. 8, 1553-60 (1994); mastalgia, drug (Drugs)32, No. 6, 477-80, (1986); prolactin-secreting pituitary tumors, journal of endocrine research (j. andrrinol. invest.)3/4, 343-347 (1980); osteoporosis, american cancer research council article (proc. annu Meet Am assoc. cancer Res.); 33: a566-7 (1992); neuroperitoneum (netroperitaneal) fibrosis, Lancet 341, No. 8841, 382 (1993).

Subtle changes in the structure of estrogen agonists can lead to significant differences in their biological properties. For example, droloxifene (3-hydroxy tamoxifen), formula I below, has 10-60 times higher affinity for estrogen receptors than tamoxifen. Droloxifene has no carcinogenic or inducing effect in vivo or in vitro, while tamoxifen causes murine liver tumors. Hasmamu et al, Cancer communication (Cancer Letter)84, 101-.

Droloxifene is reported to be effective in treating the following diseases: breast cancer, US5,047,431; endometriosis, US5,455,275; low cholesterol, US5,426,123; osteoporosis, US5254,594; prostatic hyperplasia, US5,441,986; and restenosis, US5,384,332.

The present invention provides a method of inhibiting a pathological condition selected from the group consisting of: alzheimer's disease, premenstrual syndrome, thrombomodulin deficiency, uterine fibrosis, excessive myeloperoxidase activity, hyperchrombin, autoimmune diseases, reperfusion injury of myocardial ischemia, and testosterone deficiency. The method comprises administering to a mammal suffering from said pathological condition an effective amount of a compound of formula I and optical and geometric isomers thereof; and non-toxic pharmacologically acceptable acid addition salts, N-oxides, esters and quaternary ammonium salts thereof:

wherein,

a is selected from CH₂And NR;

b, D and E are independently selected from CH and N;

y is:

(a) phenyl optionally substituted by 1-3 substituents independently selected from R⁴Substituted with the substituent(s);

(b) naphthyl optionally substituted by 1-3 independently selected R⁴Substituted with the substituent(s);

(c)C₃-C₈cycloalkyl optionally substituted by 1-2 substituents independently selected from R⁴Substituted with the substituent(s);

(d)C₃-C₈a cycloalkenyl group, a cycloalkyl group,optionally substituted by 1-2 groups independently selected from R⁴Substituted with the substituent(s);

(e) a five-membered heterocyclic ring containing a substituent selected from the group consisting of-O-, -NR-²-and-S (O)_nUp to two heteroatoms of (A) optionally substituted by 1 to 3 substituents independently selected from R⁴Substituted with the substituent(s);

(f) six-membered heterocyclic rings containing radicals selected from-O-, -NR²-and-S (O)_nUp to two heteroatoms of (A) optionally substituted by 1 to 3 substituents independently selected from R⁴Substituted with the substituent(s);

(g) bicyclic ring system obtained by condensation of a five-or six-membered heterocyclic ring containing a substituent selected from the group consisting of-O-, -NR-, and the phenyl ring²-and-S (O)_nUp to two heteroatoms of (A) optionally substituted by 1 to 3 substituents independently selected from R⁴Substituted with the substituent(s);

Z¹comprises the following steps:

(a)-(CH₂)_pW(CH₂)_q-；

(b)-O(CH₂)_pCR⁵R⁶-；

(c)-O(CH₂)_pW(CH₂)_q；

(d)-OCHR²CHR³-; or

(e)-SCHR²CHR³-；

G is: (a) -NR⁷R⁸；(b)

Wherein n is 0, 1 or 2; m is 1, 2 or 3; z²is-NH-, -O-, -S-or-CH₂-; optionally fused to one or two phenyl rings on adjacent carbons, optionally substituted independently on carbon atoms with 1-3 substituents, and optionally independently on N atom with R⁴Substituted with suitable substituents of suitable chemical nature; or

(c) Bicyclic amines containing 5 to 12 carbon atoms, either bridged or fused, and optionally independently selected from R⁴1-3 substituents of (A); or

Z¹And G may be in combination:

w is:

(a)-CH₂-；

(b)-CH＝CH-；

(c)-O-；

(d)-NR²-；

(e)-S(O)_n-；

(f)

(g)-CR²(OH)-；

(h)-CONR²-；

(i)-NR²CO-；(j)(ii) a Or

(k) -C ≡ C-; r is hydrogen or C₁-C₆An alkyl group; r²And R³Independently are: (a) hydrogen; or (b) C₁-C₄An alkyl group; r⁴The method comprises the following steps: (a) hydrogen; (b) halogen; (c) c₁-C₆An alkyl group; (d) c₁-C₄An alkoxy group; (e) c₁-C₄An acyloxy group; (f) c₁-C₄An alkyl sulfide; (g) c₁-C₄An alkylsulfinyl group; (h) c₁-C₄An alkylsulfonyl group; (i) hydroxy (C)₁-C₄) An alkyl group; (j) aryl radical (C)₁-C₄) An alkyl group; (k) -CO₂H；(l)-CN；(m)-CONHOR；(n)-SO₂NHR；(o)-NH₂；(p)C₁-C₄An alkylamino group; (q) C₁-C₄A dialkylamino group; (r) -NHSO₂；(s)-NO₂(ii) a (t) an aryl group; or

(u)-OH。

R⁵And R⁶Independently is C₁-C₈Alkyl or together form C₃-C₁₀A carbocyclic ring;

R⁷and R⁸Independently are:

(a) a phenyl group;

(b) saturated or unsaturated C₃-C₁₀A carbocyclic ring of (a);

(c) c containing up to two hetero atoms selected from-O-, -N-and-S-₃-C₁₀A heterocycle;

(d)H；

(e)C₁-C₆an alkyl group; or

(f) And R⁵Or R⁶Forming a 3-to 8-membered nitrogen-containing ring;

r, whether linear or cyclic⁷And R⁸May be arbitrarily and independently selected from C₁-C₆Alkyl, halogen, alkoxy, hydroxy and carboxy;

R⁷and R⁸The ring formed may be optionally fused with a benzene ring;

e is 0, 1 or 2;

m is 1, 2 or 3;

n is 0, 1 or 2;

p is 0, 1, 2 or 3;

q is 0, 1, 2 or 3.

Preferred compounds of the invention of formula I are of the formula:wherein G is

Or(ii) a And

R⁴is H, OH, F or Cl; and B and E are independently selected from CH and N.

Particularly preferred compounds are:

cis-6- (4-fluoro-phenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol;

(-) -cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol;

cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol;

cis-1- [6 '-pyrrolidinoethoxy-3' -pyridyl ] -2-phenyl-6-hydroxy-1, 2, 3, 4-tetrahydronaphthalene;

1- (4' -pyrrolidinoethoxyphenyl) -2- (4 "-fluorophenyl) -6-hydroxy-1, 2, 3, 4-tetrahydroisoquinoline;

cis-6- (4' -hydroxyphenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol; and

1- (4' -pyrrolidinylethoxyphenyl) -2-phenyl-6-hydroxy-1, 2, 3, 4-tetrahydroisoquinoline.

The present invention provides a method of inhibiting pathological conditions which are sensitive or partially sensitive to estrogen, antiestrogen or estrogen antagonist. They include: alzheimer's disease, premenstrual syndrome, thrombomodulin deficiency, uterine fibrosis, excessive myeloperoxidase activity, hyperchrombin, autoimmune diseases, reperfusion injury of myocardial ischemia, and testosterone deficiency.

Alzheimer's Disease (AD) is a degenerative disease of the brain characterized clinically by progressive loss of memory, cognition, rationality, judgment and emotional stability, gradually leading to severe mental disorders leading to eventual death. AD is a common cause of progressive mental disorder (dementia) in the elderly, and is considered the fourth most common medical cause of death in the united states. AD disease occurs in various races and races of the world and is a present and future public health problem. The disease is currently estimated to afflict approximately 2-3 million people in the United states alone. At present, AD has proven to be an incurable disease.

The brains of AD patients exhibit neurodegeneration and characteristic lesions, the latter of which refers to various amyloidogenic plaques, vascular amyloidosis and neurofibrillary tangles. A large number of these lesions, especially the amyloidogenic plaques and neurofibrillary tangles, are commonly found in many areas of the brain of AD patients, which are important for memory and recognition functions. A smaller number of such lesions of more restricted anatomical distribution were found in the brains of most elderly people who do not suffer from clinical AD. Amyloidogenic plaques and vascular amyloidosis are also characterized by hereditary cerebral hemorrhage with Trisomy 21(Down syndrome) and amyloidosis of the Dutch type (HCHWA-D) in the brain of an individual. Currently, the definitive diagnosis of AD often requires the discovery of the aforementioned lesions in the brain tissue of patients who die from the disease, or rarely, in small biopsy samples of brain tissue taken during invasive brain surgery.

There is a lot of evidence that the progressive cerebral deposition of a specific amyloidogenic protein, beta-amyloidogenic protein (β AP), has a seed role in the pathogenesis of AD. See Selkoe, (1991) neuron 6: 487. recently, neuronal cells grown in culture have been shown to release β AP and are present in cerebrospinal fluid in both normal humans and AD patients. See Seubert et al, (1992) Nature 359: 325-327.

Several groups (experiments) have demonstrated that one possible relevant factor for plaque pathology is direct neurotoxicity of β AP to cultured neurons. Co-therapy with TGF- β has recently been reported to reduce β AP neurotoxicity. (Chao et al, Abstract of the society of neuroscience (Soc. neurosci. abs.) 19: 1251 (1993)).

Recently, in addition to direct neurotoxicity, inflammatory responses in the brain of AD, possibly caused by β AP, have also contributed to the pathology of the disease. Limited clinical trials of the NSAID indomethacin have shown a slowing of the progression of Alzheimer's dementia (Rogers et al Science 260: 1719-1720 (1993)). European patent application 0659418a1 discloses the use of certain benzothiophenes to inhibit AD.

Despite advances in the study of the mechanism of AD, there remains a need to develop compositions and methods for treating this disease. The method of treatment should preferably be based on agents that increase the expression of TGF- β in the brain, and thus may reduce the β -amyloidogenic peptide-mediated neurotoxicity and inflammatory response associated with AD.

Several days before each month of menstruation, millions of abnormal women develop symptoms of disorganized mood and loss of appetite, which are striking similarities to those of patients with Seasonal Affective Disorder (SAD), carbohydrate-induced obesity, or bulimia nervosa. Frank refers to these syndromes as "premenstrual tension" in 1931 and is a common phenomenon. According to the Guy Abraham of UCLA, 3 or 4 of 10 patients who were in gynecological clinics suffered from premenstrual tension, and some had symptoms so severe that they had suicide impulsion. (recent Progress in Obstetrics and gynecology (Current Progress in Obstrics and Gynecology), 3: 5-39 (1980)).

The initial record of premenstrual syndrome (PMS) has focused on its association with nervous tension, headache and weight gain. The observed weight gain was initially due to excess salt and water retention, which did occur in PMS patients. But it soon proved that craving and the wide tendency to overdose of PMS patients for carbohydrates (especially sweet) was also one of the reasons. PMS is also currently known as late luteal phase syndrome (or late luteal phase disorder). (DNS, III, revision, American Psychiatric Association, 1987).

There are many statements about the etiology of PMS. For example, some have postulated that it is caused by a uterine toxin. Some believe that the common mood depression and anxiety are caused by excessive consumption of sweet food, resulting in excessive insulin secretion, too low blood sugar and insufficient brain sugar. It is also hypothesized that tissue edema causes behavioral symptoms and menstrual discomfort leading to loss and social complications leading to psychological changes.

However, these theories do not stand up: PMS remains present after hysterectomy, so uterine toxins are not the cause; hyperinsulinemia of the PMS is also not linked to hypoglycemia, and may be the result of, but not the cause of, behavioral abnormalities (i.e., the tendency of premenstrual women to select a high carbohydrate diet, which may promote insulin secretion); mood and appetite changes in the PMS are not much linked to tissue edema; non-human primates, presumably without human mental motivation or social complexity, also exhibit characteristic behavioral changes premenstrual.

There are several approaches proposed to overcome or reduce PMS symptoms. These include: diet without sugar, vitamin supplementation, ovarian hormones, antidotes, ovarian and pituitary irradiation, and the use of diuretics. However, these methods have met with only limited success.

Late luteal phase disorder (LLPDD) is a term currently associated with PMS. Many women refer to a number of physiological and psychological changes associated with specific phases of the menstrual cycle. For most of these women, the changes are less severe, cause less depression and have no impact on social and occupational functioning. In contrast, the basic feature of LLPDD is some clinically significant emotional and behavioral symptoms that are relieved within the last week of the luteal phase and within a few days after the start of the follicular phase. In most women, these symptoms occur one week before menstruation and are relieved after several days.

LLPDD is only diagnosed if the symptoms are severe enough to cause significant impairment of social or occupational function and occur during most of the menstrual cycle over the past year.

The most commonly experienced symptoms are marked mood abnormalities (e.g., sudden lacrimation, sadness, or stressful episodes), stressful persistence, anger or tension, depression, and self-mutilation. Also common are reduced interest in daily activities, fatigue and weakness, subjective feelings of inability to concentrate, altered appetite, desire to eat certain foods (especially sugar), and sleep disturbance. Other physiological symptoms may also be present, such as chest weakness or fullness, headaches, joint and muscle pain, a feeling of swelling and weight gain.

Non-steroidal anti-inflammatory drugs are typically administered to LLPDD patients, but are effective for certain physiological conditions. The physiological symptoms of PMS, if severe, can be treated symptomatically. Water retention can be alleviated by diet and urinalysis, but the severity of water retention is not always associated with psychological symptoms. Recent studies have shown that spironolactone (spironolactone, Searle) can be effective in relieving depression and crying episodes.

For other drugs, progesterone, lithium carbonate, thiazide, diuretics, antidepressants, and bronyptome (Parlodel)^RMountain soldiers) were also tested, but without success.

U.S. patent 5,389,670 describes the treatment of LLPDD/PMS with certain benzothiophenes.

In view of the deficiencies and inadequacies of existing methods of treating PMS/LLPDD, new medical approaches are needed.

The term "pre-and post-amenorrhea" refers to the period of time in a woman's life from pre-amenorrhea (reproductive age) to post-amenorrhea. This period is typically 40-60 years old, but more years before or after 50 years old. This period is characterized by rapid changes in the hormonal balance of women. Although many different hormones vary rapidly during this period, the most interesting are sex-related hormones, in particular estrogens and (to a lesser extent) progesterone. The cause of this change is the natural and time-dependent termination of ovarian function. The termination of the latter pre-and post-amenorrhea periods is marked by cessation of ovarian function or its inability to regulate the normal ovulation cycle in past women. The clinical manifestation of this end of function is a one year or longer cessation of menses. During this period (i.e., early and late phase of amenorrhea), ovarian function is continually halted, rather than a sudden or rapid event. The state before and after amenorrhea may last from about several months to more usually a year or more.

As mentioned previously, the onset and the withdrawal of amenorrhea are characterized by a change in the sex hormone composition of women, and this change has a number of sequelae. Sometimes these symptoms go past with little problem for women, but they are often the cause of moderate to severe discomfort and anxiety, and occasionally one cause of a pathological or fatal event.

It is these sequelae before and after amenorrhea that define the syndrome. Although very specific, common sequelae that result in entry into the anterior and late stages of amenorrhea include: hot secretions and perspiration, vaginal atrophy, headache, dizziness, inattention, irritability, loss of libido, arthralgia, insomnia, apathy, lassitude, muscle weakness and palpitations. ("amenorrhea" ("The Menopause"), edited by r.j. beard, Park university press, 1976, chapter 11). In addition, there is a sense of depression characterized by "amenorrhea or premenstrual syndrome". Although there is debate as to whether this is the true mental syndrome, it is a factor before and after amenorrhea ("Harrisor's principle" in medicine, "Harrison's principles of Internalmedicine", editor N.J. Isselbacher et al, 9 th edition, McGraw-Hill book Co., 1980, p.1782-. In extreme cases, some of the sequelae in some women are pathological (e.g., fluid retention and imbalance) and even fatal, especially those with a prior effect on depression. However, for most women, the main cause of discomfort and anxiety is not the occurrence of one or more of these events, but the time they must endure and their unknowns.

There is no reason to believe that any treatment reverses the aging process, so clinical treatment of the syndrome before and after amenorrhea is only palliative. Specifically, women before and after amenorrhea to be treated adopt an exogenous estrogen decline regimen. Although exogenous estrogen is effective in treating symptoms before and after amenorrhea, it does not stop the inevitable decline in ovarian function and therefore has only the effect of causing the patient to slowly enter the post-amenorrhea state. This taper therapy often requires a long period of time (in extreme cases up to several years) for ovarian function to terminate before the exogenous estrogen is discontinued. While this therapy is effective and approved, it all carries a number of side effects.

The side effects associated with estrogen therapy are not only due to estrogen but also to the concomitant progestin. In most cases, women with a uterus must administer estrogen and progesterone simultaneously or more often in a cyclical regimen. The reason for co-administration is to reduce the risk of endometrial cancer, which is likely to occur when estrogen is administered alone. Many women are unable to tolerate the effects of progesterone, resulting in a feeling of depression or even offsetting the beneficial effects of estrogen. Estrogens themselves often cause unpleasant side effects such as water retention, weight gain, overstraining, etc. Often the result is that the patient is not treated and continues to suffer from symptoms before and after amenorrhea.

Ideally, an improved therapeutic would alleviate the symptoms of the syndrome before and after amenorrhea, but would still avoid or reduce side effects. In addition, this ideal therapy can shorten the time a woman enters a stable post-menopausal state. Us patent 5,391,557 describes the treatment of premenstrual and postmenopausal syndromes, including the administration of benzothiophene compounds.

The blood clotting process, i.e. thrombosis, is due to the formation of thrombin resulting from a complex proteolytic cascade. Thrombin removes the active peptides on the a α and B β chains of fibrinogen in a manner that breaks down the proteins, which are soluble in plasma, thus starting to form insoluble fibrin.

Current anticoagulation is accomplished by administration of heparin and coumarin. Parenteral drug control of coagulation and thrombosis is the inhibition of thrombosis through the use of heparin. Heparin acts indirectly on thrombi by accelerating the inhibitory action of endogenous antithrombotic III (the major physiological inhibitor of thrombi). Heparin has not been an effective treatment because of the variable plasma level of antithrombotic III which is resistant to this indirect effect due to its surface binding. Because certain coagulation assays are believed to be associated with efficiency and safety, coagulation assays are commonly used to monitor heparin content (especially the Activated Partial Thromboplastin Time (APTT) assay). Coumarin interferes with thrombus formation by blocking post-conversion gamma-carboxylation during synthesis of prothrombin and other proteins of this type. Because of their mechanism of action, coumarins take effect very slowly, only about 6-24 hours after injection. In addition, they are not selective anti-coagulants. Coumarin also requires coagulation assay monitoring (especially thromboplastin time analysis).

For a better understanding of the present invention, the thrombin system is briefly described below. The coagulation system, sometimes referred to as "cascade" (cascade), can be thought of as a chain reaction involving the sequential activation of zymogens into active serine proteases, ultimately leading to the generation of thrombin. Thrombin converts plasma fibrinogen into insoluble fibrin by limited proteolysis. There are two important links in the coagulation cascade, the conversion of coagulation factor X to Xa by coagulation factor IXa, and the conversion of prothrombin to thrombin by coagulation factor Xa.

Both reactions occur on the cell surface, most importantly on the platelet endothelial cell surface, and both require cofactors. The major cofactors, V and VIII, circulate as relatively inactive precursors, but thrombin activates cofactors through limited proteolytic action after the initial formation of some thrombin molecules. The activated cofactors, Va and VIIIa, accelerate the conversion of prothrombin to thrombin and factor X to factor Xa by a factor of about three orders of magnitude.

Activated protein C is highly selective for two plasma protein substrates, which can be hydrolyzed and irreversibly destroyed. These plasma protein substrates are activated forms of coagulation factors V and VIII (cofactors Va and VIIIa, respectively). Activated protein C only minimally degrades inactive precursors, coagulation factors V and VIII. On dogs, it was shown that the activated protein C flash increased the circulating concentration of the major physiological fibrinolytic enzyme, tissue plasminogen activator.

However, activation of protein C involves the ultimate serine protease thrombin in coagulation cascade, and the endothelial cell membrane associated glycoprotein thrombomodulin (thrombomodulin). Thrombomodulin forms a complex with thrombin in a strictly stoichiometric ratio of 1: 1. When thrombomodulin is complexed with thrombin, the functional characteristics of thrombin are essentially modulated. On the coagulation pathway, thrombin typically coagulates fibrinogen, activates platelets, and converts coagulation cofactors V and VIII into their activated forms. Thrombin alone activates protein C but only very slowly and inefficiently. In contrast, thrombin is unable to clot fibrinogen, to activate platelets, and to convert clotting cofactors V and VIII to their activated forms in a 1: 1 complex with thrombomodulin. Thrombin: the thrombomodulin complex promotes the activation of protein C, in which thrombin: the rate constant of thrombomodulin complex activation reaction on protein C is about 20,000 times higher than that of thrombin alone.

Thus, activated protein C is an anticoagulant with a broader therapeutic range compared to other anticoagulants, such as heparin and oral hydroxycoumarin type anticoagulants like warfarin. Neither protein C nor activated protein C is effective unless thrombin is locally generated. Protein C is inactive without thrombin activation because thrombin is required to convert coagulation factors V to Va and VIII to VIIIa. It has been noted that the activated forms of these two cofactors are the preferred substrates for activating protein C. Protein C zymogen remains inactive after injection into a patient until thrombin is produced. No thrombomodulin: the thrombin complex, proenzyme protein C, is converted to active protein C at a very slow rate.

U.S. patent 5,476,862 describes the use of certain benzothiophene compounds to promote expression of thrombomodulin.

Uterine fibrosis is a long-standing and currently still present clinical problem, which has a number of names, including: uterine hypertrophy, myometrial hypertrophy, uterine fibrosis and fibrosing metritis. Uterine fibrosis is essentially a condition in which fibrous tissue is abnormally deposited in the uterine wall.

This condition can lead to dysmenorrhea and infertility in women. The precise cause of the disease is not completely understood, but there is evidence that it is due to an inappropriate response of fibrous tissue to estrogen. This condition also occurs when the mice are given daily estrogen injections for three months. This also occurs in guinea pigs when estrogen is administered daily for 4 months. The same hypertrophy also occurred in mice.

Common treatments for uterine fibrosis employ surgery, but this is expensive and sometimes causes some inconvenience, such as the occurrence of abdominal adhesions and infections. For some patients, the initial surgery is only a temporary treatment and the fibroid tissue will regenerate. In those cases, administration of a hysterectomy, although terminating fibrosis, also results in loss of fertility in women. Gonadotropins which release hormone antagonists may also be injected, but their use is limited because it can lead to osteoporosis.

U.S. patent 5,457,116 describes methods of inhibiting uterine fibrosis using certain benzothiophenes.

Autoimmune diseases involve abnormal regulation of cellular and humoral regulatory immunity and are often associated with abnormal or excessive T cell, B cell and macrophage effector function that regulates self-antigens. Activation of self-antigens by these cellular components is thought to be associated with disruption of feedback mechanisms associated with self-tolerance. Autoimmune diseases comprise a whole set of clinical disease species, which share similarities despite differences in target organs. Women of childbearing age are overwhelmingly ill, with male women having a ratio of Hashimoto's goiter of 50: 1, Systemic Lupus Erythematosus (SLE) of 10: 1, myasthenia gravis of 2: 1(Ahmed et al, J.Path., USA J.P.J.), 121: 53l (1985)). In addition, the diseases caused by the disease are all characterized as follows: chronic, clinical remission prone and unexplained episodes and involvement of other organs. Despite the appearance of autoantibodies, inappropriate expression of type II antigens, macrophage activation and T cell infiltration into target organs are all considered essentially autoimmune diseases, and the mechanism and progression of disease onset is not well understood. Thus, the treatment of these diseases is not satisfactory and involves the use of gold salts, methotrexate, antimalarials, glucocorticoids (methylprednisolone) and other immunosuppressive agents, as well as plasmapheresis and attempts to induce tolerance. Autoimmune diseases have not progressed significantly over the last decade and are primarily associated with the use of nonsteroidal and steroidal anti-inflammatory drugs to treat the symptoms of the disease. It is clear that generalized immunosuppression with glucocorticoids is prone to side effects and a greater risk of other infectious or non-infectious diseases in immunosuppressed patients when suppression of specific immune responses is necessary for the host.

Polymorphonuclear leukocytes (PMNL) play a role as regulators of inflammation. When these cells are activated, molecules that synthesize and release oxygen centers, chemoattractants, and hydrolases. This demonstrates that the oxygen center atom plays a crucial role in many diseases such as chronic inflammation, rheumatoid arthritis, SLE, etc. In autoimmune cases, such as SLE, the inflammatory response is triggered by antigenic stimulation of the patient's neutrophils or PMNL to secrete strong oxidants that destroy surrounding cells and tissues.

Although the role of estrogen in the progression and regression of autoimmune disease is very complex and depends on the nature of the disease, it appears to be relevant to autoimmune disease. For example, estrogens appear to reduce rheumatoid arthritis but exacerbate systemic lupus erythematosus (Charder & sector, annual rheumatoid disease (Ann rheum. dis.), 50: 139). As reported by Jansson (Free Rad Res. Comms.), 14(3), 195-208 (1991), incorporated herein by reference, estrogen enhances an enzyme produced by PMNL, myeloperoxidase, which regulates hydrogen peroxide to form an oxidizing agent. The enzyme converts hydrogen peroxide to the strong oxidant perchloric acid. By increasing the activity of the enzyme, perchloric acid can be generated, which increases oxidation on tissues, cells and various macromolecules of patients with chronic inflammatory/autoimmune diseases.

EP 664125A 1 reports that myeloperoxidase can be inhibited by treatment with certain 3-aroylbenzothiophenes. Excessive myeloperoxidase is associated with certain diseases including SLE, hashimoto's goiter, myasthenia gravis, rheumatoid arthritis and various sclerosis.

2-phenyl-3-aroylbenzothiophene derivatives are reported to inhibit thrombin; see EP0664126 a 1.

Estrogens have been shown to have an inhibitory effect on T cell function and an immunostimulatory response to B cells. Thus, estrogen-like compounds address the benefits associated with activated T-cells by inhibiting T-cell function, including rheumatoid arthritis, multiple sclerosis, Guillan Barre syndrome, and Hashimoto's goiter (Holmadahl, J.autoimmun.). 2: 651 (1989)).

In addition to having an inhibitory effect on T cells, estrogens also have an additional protective effect. Marui et al (J.Clin. Invest.) 92: 1866(1993)) recently reported that antioxidants inhibit the endothelial expression of VCAM-1. VCAM-1 is a ligand for VLA-4, T cells and macrophages integrin, and is involved in the translocation of these cells from the vasculature and into the spaces and target organs surrounding the vasculature. Since estrogen is an antioxidant, it is expected that estrogen and its analogs will inhibit VLA-4 dependent cell migration, thus inhibiting the immune cascade associated with autoimmune modulation diseases.

Estrogens also play a detrimental role in other autoimmune diseases, including SLE and glomerulonephritis, immune complex-related diseases. The mechanism of estrogen-regulated disease progression is unknown, and estrogen has been reported to have the ability to enhance Fc-regulated phagocytosis from estrogen-treated rodents (Flynn, Life sciences, 38: 2455(1986)) (Friedman et al, J. Clin. Res. 75: 162(1985)), and class II antigen expression and macrophage IL-1 production. These increases in macrophage-regulated effector function will likely result in immune cascades associated with self-destruction.

EP 664123A 1 reports that certain 2-phenyl-3-aroylbenzothiophenes are potent inhibitors of autoimmune diseases.

Women at pre-amenorrhea are at less risk of coronary heart disease than men of the same age; but also indicates that estrogen treatment can protect women after amenorrhea from cardiovascular diseases. Hale and Kloner have demonstrated that acute pretreatment with estradiol reduces the size of myocardial infarction caused by coronary occlusion in male and female rabbits. American journal of cool. cardio 25, 189A (1995).

In the testis and peripheral tissues of the male, a small amount of estrogen can be produced by aromatization of testosterone. Although in small quantities, usually less than one-fourth to one-tenth of that of premenopausal women, estrogens have important roles in regulating the functions of the male hypothalamus-pituitary-gonadal, skeletal development, prostate development and metabolism. In the hypothalamus, the conversion of testosterone to estrogen results in a negative feedback effect on gonadotropin releasing hormone and subsequent gonadotropin release. Estrogens therefore generally reduce the level of circulating testosterone and antagonize a corresponding increase in estrogens. As men age, the proportion of fat and lean tissue increases. Aromatization of testosterone in fat results in a gradual increase in the estrogen to testosterone ratio and an increase in negative feedback that reduces the total level of testosterone.

Gonadal hypofunction is a common occurrence in elderly men. Some studies have shown that gonadal failure may result in an observable decrease in muscle and bone weight with age. Recent studies have shown that androgen treatment can produce mild but significant improvements in muscle strength in certain gonadal-normal men. Testosterone deficiency is associated with hip fractures and bone weight is associated with testosterone levels in the elderly.

Men receiving testosterone had a significant increase in bioavailable testosterone concentration, hematocrit, right hand muscle strength, and bone calcium concentration. Their cholesterol levels were reduced (no change in HDL-cholesterol) and the BUN (blood urea nitrogen)/creatinine ratio was reduced. Morley et al, JAGS 41: 149-150(1993).

The term "preventing" is defined to include its generally accepted meaning and includes prophylactically treating a patient to prevent the occurrence of one or more of these conditions, continuously examining the symptoms of the disease, and/or treating the symptoms. Suitably, therefore, the methods of the invention encompass both medical treatment and/or prophylactic treatment.

The methods of the present invention are practiced by administering to a subject to be treated an effective amount of a compound of formula I above.

The compounds of formula I are useful for treating prostate disorders, thymus cancer, osteoporosis, endometritis, cardiovascular disease, and hypercholesterolemia, as described in commonly owned U.S. patent application 08/369,954, which is incorporated herein by reference.

The term "C₁-C₃Chloroalkyl and C₁-C₃Fluoroalkyl "includes, from one atom to all atoms substituted with chlorine or fluorine atoms to the desired degree, methyl, ethyl, propyl, and isopropyl. The term "C₅-C₇Cycloalkyl "includes cyclopentyl, cyclohexyl, and cycloheptyl.

Halogen means chlorine, bromine, iodine and fluorine. Aryl includes any of the groups independently selected from R above⁴Phenyl and naphthyl substituted with 1 to 3 substituents. DTT is dithiothreitol. DMSO denotes dimethyl sulfoxide. EDTA is ethylene diamine tetraacetic acid.

An estrogen agonist is defined herein as a compound that binds to an estrogen receptor site in mammalian tissue and can mimic the effects of estrogen in one or more tissues.

An estrogen antagonist is defined herein as a compound that binds to an estrogen receptor site in mammalian tissue and prevents estrogenic effects in one or more tissues.

It will be understood by those of ordinary skill in the art that certain of the substituents listed herein may be incompatible with each other or with heteroatoms in the compounds, and that such incompatibility should be avoided in the selection of the compounds of the present invention. He should also recognize that certain functional groups require protecting groups during synthetic procedures.

One of ordinary skill in the art will recognize that certain compounds of the present invention will contain atoms having particular optical or geometric configurations. The present invention encompasses all such isomers; the typical levorotatory isomer having the cis configuration is preferred. Likewise, the skilled artisan will recognize that a variety of pharmaceutically acceptable salts and esters may be prepared from certain compounds of the invention. All such esters and salts are included in the present invention.

The drugs for diseases or conditions of the present invention can be prepared by a conventional method using conventional organic or inorganic additives, for example, excipients (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate), binders (e.g., cellulose, methyl cellulose, hydroxymethyl cellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, acacia, polyethylene glycol, sucrose or starch), disintegrators (e.g., starch, carboxymethyl cellulose, hydroxypropyl starch, lower-substituted hydroxypropyl cellulose, sodium bicarbonate, calcium phosphate or calcium citrate), lubricants (e.g., magnesium stearate, light anhydrous silicic acid, talc or sodium lauryl sulfate), flavors (e.g., citric acid, methanol, glycine or orange powder), preservatives (e.g., sodium benzoate, sodium bisulfite, sodium hydrogen sulfite, calcium phosphate or calcium carbonate), lubricants (e.g., gelatin, sodium hydroxide, sodium lauryl sulfate, sodium hydroxide, Methyl or propyl parabens), stabilizers (such as citric acid, sodium citrate or acetic acid), suspending agents (such as methylcellulose, polyvinylpyrrolidone or aluminum stearate), dispersing agents (such as hydroxypropylmethylcellulose), diluents (such as water) and base waxes (such as cocoa butter, white petrolatum or polyethylene glycol). In pharmaceutical compositions, the active ingredient may be present in an amount to achieve the desired therapeutic effect, for example, in a single dose of 0.1 to 50mg for oral and parenteral administration.

The active ingredient is contained in an amount of 0.1 to 50mg per single dose, and is usually administered to a patient one to four times per day, but the above dose may be suitably changed depending on the age, body weight, condition and administration form of the patient. The preferred dosage is 0.25-25 mg. Preferably one dose per day.

The compounds of the present invention can be readily prepared by the following schemes.

Certain compounds of formula I may be conveniently hydrogenated with a noble metal catalyst in a reaction-inert solvent to form a compound of formula (II):

to an unsaturated intermediate of (a); the pressure and temperature are not critical, and the hydrogenation is generally in the range of 20 to 80 lbs/inch²Hydrogen pressure at room temperature for several hours.

If desired, the hydrogenation product is isolated, purified and the ether groups are decomposed using an acidic catalyst in a reaction-inert solvent at a temperature of about 0 ℃ to 100 ℃ depending on the acidic catalyst used. HBr at high temperatures, BBr from 0 ℃ to ambient temperature was found₃And AlCl₃This reaction is effective.

The product, a compound of formula I, is isolated and purified using standard methods.

Wherein A is CH₂And B, D and E are CH are described in U.S. patent No. 3,274,213; journal of medicinal chemistry (j.med.chem.)10, 78 (1967); journal of pharmaceutical chemistry 10, 138 (1967); and journal of pharmaceutical chemistry 12, 881(1969), the disclosure of which is incorporated herein by reference. They can also be prepared by the following methods.

Scheme 1 represents the preparation of compounds of formula I wherein e ═ 1 and a ═ CH₂，Z¹＝OCH₂CH₂G ═ cycloalkylamine, B ═ CH. Alkylation of 4-bromophenol with the corresponding N-chloroethylamine in a polar aprotic solvent such as dimethylformamide using potassium carbonate as a base at elevated temperature affords compounds 1-2, where D and E are CH. The preferred temperature is 100 ℃. The synthesis of the compound 1-2 of which D or E or both are N is completed by the nucleophilic substitution reaction of hydroxyethyl cyclylamine on a dibromo compound (1-1) to generate bromamine (1-2) under the condition of phase transfer. Synthesis (Synthesis), 77, 573 (1980). Followed by metallation with n-butyllithium or magnesium metalAfter halide exchange, the bromoamine (1-2) forms the corresponding lithium or magnesium reactant which is reacted at low temperature, in the presence of strontium chloride, preferably (as usual without strontium chloride) with 6-methoxy-1-tetralone to form methanol (derivative) (1-3) or styrene (1-4) after acid reaction. Treatment of methanol (1-3) or styrene (1-4) with a brominating agent such as pyridinium bromide affords bromostyrene (1-5). Aryl or heteroaryl zinc chloride or aryl or heteroaryl bromic acid is reacted with the bromide of (1-5) in the presence of a palladium metal catalyst such as tetrakis (triphenylphosphine) palladium (0) to produce diarylstyrene (1-6). [ theory and applied chemistry (Pure)&Applied chem.63, 419, (1991) and ball. chem.Soc. Jpn. (Japanese chemical Association bulletin) 61, 3008-3010, (1988)]. To prepare the preferred compounds, substituted phenylzinc chloride or substituted phenylbromic acid is used in the reaction. The preparation of aryl zinc chloride is obtained by quenching the corresponding lithium reactant with anhydrous zinc chloride. Aryl bromic acids, which are not commercially available, can be prepared by quenching the corresponding aryl lithium reactant with a trialkyl bromate (preferably trimethyl or triisopropyl bromate) followed by an acid treatment. Acta Chemica Scan, 47, 221-230 (1993). Commercially unavailable lithium reactants can be prepared by halogen metal exchange of the corresponding bromide or halide with n-butyl or t-butyl lithium. In addition, lithium reactants can also be prepared from heteroatom-assisted lithiation Reactions (lithiations) described in "Organic Reactions", volume 27, chapter i. Catalytic hydrogenation of 1-6 in the presence of palladium hydroxide, for example on activated carbon, gives the corresponding dihydromethoxy intermediate, followed by demethylation with boron tribromide in dichloromethane at 0 ℃ or hydrogen bromide in 45% acetic acid at 80-100 ℃ gives the compound of (1-7). These compounds are racemic and can be resolved into enantiomers by high pressure liquid chromatography using a column of chiral stationary phase, e.g., a Chiralcel OD column. In addition, optically pure salts, such as diastereomeric salts formed with 1, 1 '-dinaphthyl-2, 2' -diyl hydrogen phosphate, are recrystallized to complete the optical resolution.

The cis-structure of the compound (1-7) may be treated with a base to the trans-structure.

When D and/or E is nitrogen, the intermediates (formula II) and compounds of formula I can be prepared from the corresponding dihalopyridines or pyrimidines as shown in scheme 1, and example 6 details the preparation of 6-phenyl-5- [6- (2-pyrrolidin-1-yl-ethoxy) pyridin-3-yl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol.

Wherein e is 1, A is CH₂，Z′＝OCH₂CH₂Methyl ethers of compounds of formula I, G ═ pyrrolidinyl, D, E, B ═ CH, Y ═ Ph, can be conveniently prepared as follows: in an inert reaction solvent in the presence of a noble metal catalyst, a first step of reaction of naphthyridine (Upjohn)&Co., 700 Portage Road, Kalamazoo, Michigan 49001). Pressure and temperature requirements are not critical, room temperature in ethanol and 50 lb/in²The reaction is easily carried out for about 20 hours under the pressure of (1).

The second step is the decomposition of the methoxy group, which can be easily accomplished at room temperature with an acidic catalyst such as boron tribromide in a reaction-inert solvent or with hydrogen bromide in acetic acid at 80-100 ℃. The product is isolated by conventional means and converted to an acidic salt if desired.

Compounds of formula I wherein B is nitrogen are prepared by the methods of schemes 2 and 3, and are described in examples 3-5 and 10-12.

The preparation of compounds of formula I where B ═ N is shown in scheme 2. Aryl acid chloride (2-1) is treated with a primary amine to give the aryl secondary amide (2-2), followed by reduction to secondary amine (2-3) with lithium aluminum hydride in diethyl ether solvent. Then, acylation is carried out with aroyl chloride to obtain tertiary amine (2-4), and cyclization is carried out under the action of hot phosphorus oxychloride to generate dihydroisoquinolinium salt (2-5). Reducing sodium borohydride to generate alkoxy tetrahydroisoquinoline, and then removing methyl by using boron tribromide in dichloromethane to obtain the target compound.

The synthesis of compounds of formula I, wherein B ═ N, is also depicted in scheme 3. Acylation of secondary amine (3-1) with benzyloxyaroyl chloride (3-2) affords tertiary amine (3-3), cyclization with hot phosphorus oxychloride to yield dihydroisoquinoline salt (3-4). Reduction of (3-4) with sodium borohydride followed by debenzylation with aqueous hydrochloric acid to yield isoquinoline(3-5) which is further alkylated with a suitable functionalized chloride and demethylated with boron tribromide to afford the target compound. Scheme 2

In each flow chart, the following symbols have the following meanings: ar: aryl Ph: phenyl PYR HBrBr₂: pyridinium perbromide.

Scheme 1

Scheme 3

Although the free base form of the compounds of formula I may be used in the process of the present invention, it is preferred to prepare and use a pharmaceutically acceptable salt form. Thus, the compounds used in the process of the invention form pharmaceutically acceptable acid and base addition salts, the acids being various inorganic acids, preferably organic acids, and including physiologically acceptable salts commonly used in pharmaceutical chemistry. These salts are also part of the present invention. The mineral acids used to form this salt include: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, hypophosphorous acid, and the like. Compounds derived from organic acids, such as aliphatic mono-or dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids and hydroxyalkanol diacids, aromatic acids, aliphatic and aromatic sulfonic acids, may also be used. Thus, such pharmaceutically acceptable salts include: acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, beta-hydroxybutyric acid, butyne-1, 4-dioate, hexyne-1, 4-diolate, hexanoate, octanoate, chloride, cinnamate, citrate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, benzoate, maleate, hydroxymaleate, malonate, mandelate, methanesulfonate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, benzoate, dihydrogenate, dihydrogenbutyrate, dihydrogen, Phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, propiolates, propionates, phenylpropionates, salicylates, sebacates, succinates, suberates, sulfates, hydrogensulfates, pyrosulfates, sulfites, bisulfites, sulfonates, benzenesulfonates, p-bromobenzenesulfonates, chlorobenzenesulfonates, ethanesulfonates, 2-hydroxyethanesulfonates, methanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, p-toluenesulfonates, xylenesulfonates, tartrates, and the like. A preferred salt is citrate.

Pharmaceutically acceptable acid addition salts are generally prepared by reacting a compound of formula I with an equimolar or excess amount of acid. The reactants are typically combined in a mutual solvent such as diethyl ether or benzene. The salt generally precipitates from solution in one hour to 10 days and can be isolated by filtration or the solvent removed by conventional methods.

The pharmaceutically acceptable salts of the compounds of formula I generally have better solubility properties than the parent compound and are therefore easier to formulate in liquid or emulsion form.

Once prepared, the free base or salt form of the compound of formula I may be administered to the subject in need thereof by the methods described herein. The following non-limiting test examples illustrate the process of the present invention.

In the methods of the invention, the compound of formula I is administered continuously or from 1 to 4 times a day.

The term "effective amount" as used herein refers to an amount of a compound of the present invention that inhibits the manifestation of the pathological condition herein. The specific dose of the compound to be administered according to the present invention is determined in accordance with the specific case, for example, the compound to be administered, the mode of administration, the condition of the patient and the severity of the pathological condition to be treated. Typical daily dosages contain non-toxic dosage levels of from about 0.25mg to about 100mg per day of a compound of the present invention. A preferred daily dosage is from about 10mg to about 40mg per day.

The compounds of the present invention may be administered via a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. The compounds are preferably formulated prior to administration, the choice being determined by the clinician. Typically, a compound of formula I or a pharmaceutically-acceptable salt thereof is combined with a pharmaceutically-acceptable carrier, diluent or excipient to form a pharmaceutical formulation.

The total active ingredients in such a formulation are from 0.1% to 99.9% of the total formulation. By "pharmaceutically acceptable" is meant that the carrier, diluent, excipient, and/or salt must be compatible with the other ingredients of the formulation and not deleterious to the patient.

Pharmaceutical formulations containing the compounds of formula I may be prepared using methods well known in the art and well known readily available ingredients. For example, the compounds of formula I may be formulated with common excipients, diluents or carriers and formulated into tablets, capsules, suspensions, powders and the like. Examples of carriers, excipients and diluents suitable for this formulation include fillers and extenders such as starches, sucrose, mannitol and silicic acid derivative binders (e.g., carboxymethyl cellulose and other cellulose derivatives), alginates, gelatin and polyvinyl pyrrolidone; humectants such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate, dissolution retarding agents such as paraffin; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol and glycerol monostearate, absorptive carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, and solid polyethylene glycols.

The compounds may be formulated as elixirs or solutions for convenient oral administration or as solutions suitable for parenteral administration, for example for intramuscular, subcutaneous or intravenous administration.

In addition, the compound is very suitable for preparing sustained-release preparations and the like. The formulation is such that it may release the active ingredient alone or, preferably, may release at a particular physiological site over an extended period of time. The covering, containment and protective matrix may be made of, for example, a polymer or wax.

The compounds of formula I are generally administered in a convenient formulation. The following formulation examples are intended to be illustrative only and are not intended to limit the scope of the present invention.

In the following formulations, "active ingredient" refers to a compound of formula I or a salt thereof.

Preparation 1: gelatin capsule

Hard gelatin capsules were prepared as follows:

ingredient amount (mg/capsule)

0.25-100% of active ingredient

Starch, NF (national drug group) 0-650

Starch flowable powder 0-50

Siloxane fluid, 350 centistokes 0-15

A tablet formulation prepared using these ingredients was as follows:

preparation 2: tablet formulation

Component amount (mg/tablet)

0.25-100% of active ingredient

Cellulose, microcrystalline 200-

Silicon dioxide, foamed 10-650

Stearic acid 5-15

These components are mixed and compressed into tablets.

In addition, tablets containing 0.25-100mg of active ingredient per tablet are prepared as follows:

preparation 3: tablet formulation

Component amount (mg/tablet)

0.25-100% of active ingredient

Starch 45

Cellulose, microcrystalline 35

Polyvinylpyrrolidone 4

(10% aqueous solution)

Sodium carboxymethylcellulose 4.5

Magnesium stearate 0.5

Talc powder 1

The active ingredient, starch and cellulose were filtered through a 45 mesh U.S. sieve (No.45 mesh u.s.sieve) and mixed thoroughly. Polyvinylpyrrolidone was mixed with the resulting powder and then filtered through a 14 mesh U.S. sieve. The granules thus obtained are dried at 50-60 ℃ and filtered through a No. 18 mesh U.S. sieve. The sodium carboxymethylcellulose, magnesium stearate and talc are filtered through a 60 mesh U.S. sieve, then added to the granules, mixed and compressed into tablets on a tablet press.

A unit suspension containing 0.25-100mg of active ingredient per 5ml of medicament is prepared as follows:

preparation 4: suspending agent

Amount of ingredients (mg/5ml)

0.25-100mg of active ingredient

Sodium carboxymethylcellulose 50mg

Syrup 1.25mg

Benzoic acid solution 0.10ml

The flavoring agent is in sufficient quantity

A sufficient amount of colorant

Purified water to 5ml

The dosage form was filtered through a 45 mesh U.S. sieve and mixed with sodium carboxymethylcellulose and syrup to form a slip paste. Benzoic acid diluted with water, flavouring and colouring agents were added with stirring. Sufficient water was then added to bring the volume to the desired volume. An aerosol was prepared containing the following ingredients:

preparation 5: aerosol and method of making

Component amount (% by weight)

Active ingredient 0.25

Ethanol 25.75

Propellant 22 (chlorodifluoromethane) 70

The active ingredient is mixed with ethanol, the mixture is added to a portion of propellant 22, cooled to 30 ℃ and transferred to a filling unit. The desired amount was then added to a stainless steel container and diluted with the remaining propellant. The valve member is then installed into the container.

The suppository is prepared as follows:

preparation 6: suppository

Component amount (mg/suppository)

Active ingredient 250

Saturated fatty acid glyceride 2000

The active ingredient is filtered through a 60 mesh U.S. sieve and suspended in saturated fatty acid glycerides that have to be melted thermally in a pre-use minimum. The mixture is then poured into a plug, typically of 2 gram capacity, and cooled.

The formulation for intravenous use was prepared as follows:

preparation 7: intravenous solution

Amount of the ingredients

20mg of active ingredient

Isotonic saline 1,000ml

The solution of the above components is intravenously injected into the patient at a rate of about one minute to one milliliter.

Detection of inhibition of Alzheimer's disease

EP 0659418a1 describes the detection of compounds that are effective in the treatment of AD.

Amylopectin is available from Bachem Inc (Torrance, ca), Peninsula Laboratories, Inc (Belmont, ca), and Sigma Chemicals, st. Amyloid-beta (1-40) and anti-beta-amyloid peptide (40-1) are available from bachem. Beta is a₂Microglobulin is available from Sigma Chemicals (st. louis, missouri).

Peptide stock solutions (1mM) were prepared fresh in pyrogen-free sterile water and diluted to the indicated concentrations in the required medium. Mouse hippocampal cultures (10-14 days in vitro) were treated with peptide or vehicle for four days. Survival of murine cortical cultures was judged by comparative microscopy observations and quantified by measuring the release of Lactate Dehydrogenase (LDH) into the culture medium.

Detection 1

Primary mouse hippocampal neurons were cultured in vitro using standard cell culture techniques. Amyloid- β (a β) peptide was added to the cultured cells at a normal toxic concentration of 25 μm. Viability was assessed 4 days after treatment by measuring LDH released into the medium. LDH was measured in aliquots of 96-well plates in standard 340nm kinetic LDH assays (Sigma catalog #228-20) in DMEM at 20 μ l volumes. The analysis was performed at 37 ℃ on a PC-driven EL 340 microdisk biokinetics (Microplate Biokinetics) disk reader (Bio-Tek instrument) with data analysis using Delta Soft II software (version 3.30B, BioMetallics, Inc.). Each assay was run with quality control standards containing standard and elevated serum LDH levels (e.g., Sigma enzyme control groups 2N and 2E). Results are expressed in LDH/liter, where 1 unit is defined as the amount of enzyme required to catalyze the production of 1 micromole of coenzyme I per minute under the assay conditions. For protection studies, a compound of formula I was added to the culture prior to and/or simultaneously with treatment with a β.

The activity of the compound of formula I can be demonstrated by a reduction in LDH released into the medium (neurotoxicity indicator) relative to the control group.

Detection 2

Four vascular closures were performed on 5-15 mice for 15 minutes resulting in systemic ischemia. The compounds of the invention are administered to experimental and control animals before, simultaneously with and/or after 15 minutes after closure. Animals were sacrificed 3 days after ischemic insult and neuronal damage to the hippocampus and striatum was assessed by standard histological observations.

The activity of the compounds of formula I is illustrated by a reduction in neuronal damage.

Detection 3

5-15 women were selected for clinical study. Women were post-amenorrhea, i.e. had stopped menstruation for 6-12 months before the study began, they were diagnosed with early AD and expected to have worsening AD symptoms during the study, but otherwise in good health. Placebo-controlled groups, i.e., women, were studied in two groups, one group receiving a compound of the invention and the other group receiving a placebo. Patient memory, recognition, thrust and other symptoms associated with AD are benchmarked. Women in the experimental group took 10-100mg of active substance per day. They continued to be treated for 6-36 months. The baseline-labeled symptoms were recorded accurately for both groups and the results were compared at the end of the study. Results were compared not only between members of each group, but also for each patient with symptoms reported for each patient before the study began. The activity of the tested drugs is indicated by a reduction in the typical cognitive decline and/or behavioral disorders associated with AD.

The activity of at least one of the above assays may demonstrate the utility of the compounds of formula I.

PMS/LLPDD test method

3-15 women were selected for clinical study. Women have normal menstruation, good health, and one or more of the above PMS/LLPDD symptoms. Because these symptoms have idiosyncratic and subjective characteristics, placebo-controlled groups were also studied, i.e., women were divided into two groups, one group receiving the active agent of the invention and the other group receiving a placebo. Women in the experimental group took 10-100mg of the drug orally daily. They continued to be treated for 1 to 3 months. The number and extent of symptoms in both groups were accurately recorded and the results were compared at the end of the study. Results were compared not only between members of each group, but also for each patient with the reported symptoms of each patient before the study began. See us patent 5,389,670.

The utility of the compounds of the invention in inhibiting the symptoms of PMS/LLPDD can be demonstrated by using the positive effects on one or more symptoms studied as above.

Experimental method for inhibiting premenstrual syndrome of amenorrhea experiment 1

A group of 3-20 women of 40-50 years old was selected as the experimental group. Women all exhibit at least one condition associated with impending amenorrhea. The compounds of the invention are administered at a dose of 10-100 mg/day and the development of symptoms is closely monitored. The compound of the invention was administered for 3 weeks. Experiment 2

The same experiment as experiment 1 was performed, but the treatment administration time was 3 months. Experiment 3

The same experiment as experiment 1 was performed except that the administration time was 6 months.

"Activity" is defined herein as the complete cessation or alleviation or nonoccurrence of one or more symptoms or the rapid onset of the post-amenorrhea state in a patient, each of which indicates that the compounds of the invention are effective in treating the pre-and post-amenorrhea syndrome.

Detection of increased thrombomodulin expression

The following assays are described in EP0659427 and US 5476862, incorporated herein by reference.

Detection 1

To further understand the effects of the compounds of formula I, the intimal smooth muscle cells and its role in enhancing blood anticoagulation, it is necessary to investigate changes in Thrombomodulin (TM) activity at the surface of these cells. The compounds of formula I may also be used to reverse/correct the effects of modulators that decrease TM activity on the cell surface.

Approximately 40,000 and 80,000 early inserted endothelium (arterial, venous, or micro-subcutaneous), or intimal smooth muscle cells were seeded and grown into fusions on 24-well cell culture plates. The cell monolayer was washed sequentially 2-3 times with Hank buffered saline (HBSS) or Serum Free Medium (SFM). Various concentrations of the compound of formula I (ranging from micromolar to picomolar) were added to the cells in triplicate over 24 hours. Cells in negative control wells were maintained in serum-free medium and equal amounts of vehicle were given in all wells.

The current method of measuring cell surface TM activity is to use a two-step amidolytic assay. In the first step of the assay, HBSS or SFM washes, 0.4ml SFM containing human protein C (final concentration 11.2. mu.g/ml) and human alpha-coagulationEnzyme (final concentration 0.1 NIHU/ml) was added to the monolayer and incubated at 37 deg.C and 5% CO₂And (4) carrying out incubation. At time points 15, 30 and 45 minutes, 100 μm of medium was removed from each well and 50 μ l of excess hirudin was added to the microwell at 37 ℃ for about 5 minutes in order to further inhibit thrombin activity. As described above, a mixture of SFM and protein C and alpha-thrombin in the absence of cells was used as a negative control and similarly treated.

In the second step of the assay, 3mM 2366 of chromogenic substrate for protein C, approximately 50. mu.l, are added to the conditioned culture/hirudin mixture and OD 405 is measured using an automatic plate reader to monitor the kinetic mechanism of TM activity within 4 minutes. Upon completion of this kinetic analysis, total protein was measured using the BCA method. The final TM activity is expressed as% increase.

Detection 2

The babon model of E-Coil induced sepsis as described in US 5009889 (incorporated herein by reference) was used to demonstrate the efficacy of compounds of formula I as antithrombotic agents and their ability to modulate inflammation-induced endothelial dysfunction.

The utility of the compounds of the present invention can be confirmed by the following results shown in any of the above assays: positive effects on thrombomodulin expression, coagulant dysregulation, or protein C activation rate characteristics.

Experiment on inhibition of uterine fibrosis

Experiment 1

3-20 women suffering from uterine fibrosis were administered the compound of the present invention. The dosage of the compound is 0.1-100 mg/day, and the administration period is 3 months.

These women were observed during the dosing period and three months after drug withdrawal to see the effect on uterine fibrosis.

Experiment 2

Same as experiment 1, but the administration period was 6 months.

Experiment 3

Same as experiment 1, but the administration period was 1 year.

Experiment 4

A. Induction of fibroadenomas in guinea pigs

In sexually mature female guinea pigs, leiomyoma induction was induced by long-term estrogen stimulation. The animals are injected with estradiol 3-5 times per week for about 2-4 months or until tumors develop. Treatment involves administering a compound or vehicle of the invention for about 3-16 weeks, then the animals are sacrificed and the uterus is extracted for analysis of tumor regression.

B. Transplanting human uterine fibrous tissue into nude mice

Leiomyomas from humans are transplanted into the peritoneal cavity and/or myometrium of sexually mature, castrated female nude mice. Exogenous estrogen is injected to induce growth of the transplanted tissue. In some instances, the collected tumor cells are cultured in vitro in a transplant precursor. Treatment included gastric lavage with daily treatment of the compound or excipient of the invention for 3-16 weeks, and the grafts were removed and measured for growth or regression. At sacrifice, the uterus was harvested to assess the condition of the organ.

Detection 5

A. Uterine fibroids from humans were harvested and maintained in vitro as a primary non-deforming culture. The surgical sample is passed through a sterile mesh or sieve or otherwise removed from the surrounding tissue to prepare a single cell suspension. Cells were maintained in medium containing 10% serum and antibiotic agents. The growth rate in the presence or absence of estrogen is determined. The cells were analyzed for their ability to produce the supplemental component C3 and for their response to growth factors and growth hormones. The cultures were analyzed in vitro for their reproductive response after treatment with progesterone, GnRH, a compound of the invention and vehicle. Levels of steroid hormone receptors were analyzed weekly to determine if important cellular features were maintained in vitro. Tissues from 5-25 patients were used.

At least one more analytical activity confirms that the compounds of the invention are capable of treating uterine fibrosis.

Confirmation of detection of myeloperoxidase inhibition

Detection 1

To investigate the properties of the compounds of formula I in inhibiting myeloperoxidase activity, assays 1 and 2 described by Jansson (Supra.) were used.

In this assay, human PMN platelets were stimulated with estriol in the presence of additional hydrogen peroxide to increase myeloperoxidase activity. Chemiluminescence measures the conversion of luminol with perchloric acid. The reaction mixture contains cells (10)⁶) Reagent or compound of formula I (1 μm), hydrogen peroxide (0.1mM), and luminol (0.2mM), incubated at 37 ℃.

Estrogens and their analogs stimulate myeloperoxidase activity. The compounds of formula I antagonize estriol-stimulated chemiluminescence.

Detection 2

The purified human myeloperoxidase was incubated with estrogen or the compound of formula I in the presence of luminol at 37 ℃. Substrate hydrogen peroxide was added and chemiluminescence was measured. The reaction mixture was human MPE (250ng), reagent or compound of formula I (10 μm, titrated), hydrogen peroxide (1mm) and luminol (0.2 mm).

Estrogens and their analogs have little or no effect on MPE activity, but the compounds of formula I reduce the activity of purified MPE.

Detection 3

5-15 women were selected for clinical study. Women suffer from SLE or rheumatoid arthritis. Because of the idiosyncratic and subjective nature of these diseases, placebo-controlled groups were studied, i.e. women were divided into two groups, one of which received the compound of formula I as the active substance and the other of which received a placebo. The experimental group women take 50-100 mg of the medicine orally every day. The treatment lasts for 3-12 months. The number and extent of symptoms in both groups were kept accurately documented and the results compared at the end of the study. Not only were the results compared between the two groups of members, but the results for each patient were also compared to the reported symptoms for each patient prior to the study initiation.

The utility of the compounds of formula I can be demonstrated by the positive effect of at least one of the tests described above.

Indicating the detection of inhibition of Thrombin

method-t-P_AEffect on dissolution of human plasma clot

Human plasma clots were formed in microtubes as follows: add 50. mu.l thrombin (73NIH units/ml) to 100. mu.l human serum containing 0.0229. mu. Ci¹²⁵I labeled fibrinogen. Lump dissolution was studied by smearing the lumps with 50. mu.l urokinase or streptokinase (50, 100 or 1000 units/ml) and incubation at room temperature for 20 hours. After incubation, the tubes were centrifuged in a Beckman Microfuge. 25ml of the upper liquid layer was added to 1.0ml of 0.03M tris (tromethamine)/0.15M NaCl buffer for gamma-counting. The thrombin (and alternate buffer) was omitted to give a count of 100% dissolved controls. The possible interaction of thrombin inhibitor with fibrinolysis was estimated by adding the compounds to the upper solution (concentrations 1,5 and 10. mu.g/ml). For a particular concentration of fibrinogen solubilization, the IC was approximated by extrapolating from some data points to a value representing 50% solubilization by linear extrapolation₅₀The value is obtained.

Anti-coagulation Activity

Material

Dog and mouse plasma were obtained from awake hybrid puppies (unlimited, Hazelton-LRE Kalamazoo, Mich., USA) or anesthetized male Sprague-Dawley mice (Harlan Sprague-Dawley, Inc., Indianapolis, Ind. Napolis, USA) by intravenous injection of 3.8% citrate. Fibrinogen was prepared as fractions 1-2 from instant ACD (citric acid dextrose) human blood according to previous methods and instructions. Smith, journal of biochemistry, 185, 1-11 (1980); and Smith et al, biochemistry (biochem. J.), 11, 2958-2967 (1972). Human fibrinogen, 98% pure/plasmaseless, was purchased from American diagnostic, grenningham, ct, usa. The coagulant ACTIN thromboplastin and human plasma were obtained from Baxter Healthcare Corp, Dade Division, Miami, Florida, USA. Bovine thrombin from Parke-Davis (Ann Detroit, Mich.) was used in plasma for coagulation assays.

Method

Measurement of anti-coagulation Properties

Coagulation assay methods As previously described, Smith et al, Thrombus research, 50, 163-174 (1988). A coascaner coagulometer (American LABor, Inc.) was used to perform all coagulation tests. Thromboplastin time (PT) was measured by adding 0.05ml saline and 0.05ml thromboplastin-C reagent to 0.05ml test plasma. 0.05ml CaCl was added after incubation of 0.05ml test plasma with 0.05ml Actin reagent for 120 seconds₂(0.02M) to measure thromboplastin time of the activated moiety (APTT). Thrombin Time (TT) was measured by adding 0.05ml saline and 0.05ml thrombin (10NIH units/ml) to 0.05ml test plasma. The compounds of formula I are added to the plasma of humans or animals at a wide range of concentrations to determine the prolonged effect on APTT, PT and TT assays. Linear extrapolation is used to estimate the concentration that requires doubling the clotting time for each test.

Animal(s) production

Male Sprague Dawley mice (350-425 gm, Harlan Sprague Dawley, Inc. Frdianapolis, Ind.) were anesthetized with xylazine (20mg/kg, subcutaneous) and ketamine (120mg/kg, subcutaneous) and placed on a heated water blanket (37 ℃). The cannula is inserted into the jugular vein for infusion.

Artery-vein bypass model

The left and right jugular veins were inserted into a 20 cm long polyethylene PE 60 tube. The central area of a 6cm larger tube (PE 190) with cotton thread (5cm) in the lumen was friction fitted between the longer areas to complete the arterial venous bypass circuit. Blood was circulated in the bypass for 15 minutes, after which the cotton thread was carefully removed and weighed. The weight of the wet cotton thread was reduced from the total weight of cotton thread and thrombus (see j.r. smith, british journal of pharmacology (br.j. pharmacol.), 77: 29, 1982).

FeCl of arterial injury₃Model (model)

The carotid artery was isolated by a midline ventral cervical incision. A thermocouple was placed under each artery and the vessel temperature was continuously recorded on a bar chart. A tube sleeve (0.058 ID × 0.077 OD × 4mm, Baxter medical grade Silicone) was cut longitudinally and placed around each carotid artery directly above the thermocouple. FeCl₃·6H₂O dissolved in water at a concentration (20%) of FeCl₃Is shown in real concentration. To damage the artery and initiate thrombosis, 2.85 μ l were pipetted into the tube to wash the artery on the thermocouple probe. A sharp decrease in temperature indicates arterial occlusion. Closure time is expressed in minutes and represents the application of FeCl₃And the time elapsed between a rapid decrease in blood vessel temperature (see k.d. kurz, thrombi study (Thromb. Res.), 60: 269, 1990).

Spontaneous thrombosis model

In vitro data show that peptide thrombin inhibitors inhibit thrombin and other serine proteases, e.g., plasmase and plasmase pro-activators. To assess whether the compounds inhibit fibrinolysis in vivo, the rate of spontaneous thrombus formation was determined by implanting labeled whole blood clots into the pulmonary circulation. Mouse blood (1ml) with calf thrombin (4IU, Parke Davis) and¹²⁵fibrinogen (5. mu. Ci, ICN) was mixed rapidly, immediately injected into an elastic tube and incubated at 37 ℃ for 1 hour. The aged thrombus was removed from the tube, cut into 1cm pieces, washed 3 times in normal saline, and each piece was counted on a gamma counter. A segment of known count is aspirated into the catheter and then implanted into the jugular vein. The catheter tip enters near the right atrium and expels the clot to drift through the pulmonary circulation. After 1 hour of transplantation, the heart and lungs were collected and counted separately. Dissolve thrombus

Fibrinolysis of the implant clumps is a function of time. (see, J.P. Clozel, cardiovascular pharmacology, 12: 520, 1988).

Coagulation parameter

Plasma Thrombin Time (TT) and Activated Partial Thrombin Time (APTT) were measured with a fibrometer. Blood was sampled from the cervical catheter and collected in syringes containing sodium citrate (3.8%, 1 part versus 9 parts blood). To measure TT, murine plasma (0.1ml) was mixed with saline (0.1ml) and bovine thrombin (0.1ml, 30U/ml in Tris buffer, Parke Davis) at 37 ℃. For APTT, plasma (0.1ml) and APTT solution (0.2. mu.l, Organon Teknika) were incubated at 37 ℃ for 5 minutes, CaCl was added₂(0.025M) to start caking. The test was repeated twice and averaged.

Bioavailability index

Assuming that the increase in TT is due solely to thrombin inhibition by the parent compound, a measure of biological activity, TT, is used as an alternative to detecting the parent compound. The time period of thrombin inhibitor effect on TT was determined after intravenous bolus injection in anesthetized mice and oral treatment of fasted conscious mice. Because of the limitation in blood volume and the limitation in the number of points needed to determine the time period from treatment to when the response returned to pre-treatment values, two groups of mice were used. Each experimental member represents alternating sequential time points, and the area under the curve (AUC) was calculated using the average TT over the time period. The biological activity index is calculated as follows and expressed as a percentage of relative activity. The area under the curve (AUC) of the plasma TT time period was measured and adjusted according to the dose. This bioavailability parameter is expressed in "% relative activity" and calculated as follows:

compound (I)

Compound solutions were prepared fresh daily in normal saline and injected as a bolus or 15 minutes prior to experimental perturbations or throughout. The bolus injection volume was 1ml/kg intravenously, 5ml/kg orally, and the injection volume was 3 ml/hr.

Statistics of

Results are presented as mean +/-SEM. Statistical significance differences were measured using one-way difference analysis, and then Dunnett's experiments were used to determine which mean values were different. The significance level of the ineffective precondition of rounding off the same average value is P < 0.05.

Animal(s) production

Male dogs (Beagles, 18 months-2 years; 12-13kg, Morshall Farms, North Rose, New York 14516) fasted overnight and were fed Purira approved prescription foods (Purina Mills, St. Louis, Mo.) for 240 minutes after dosing. The water can be drunk at will. Keeping the temperature at 66-74 ℃ F; 45-50% relative humidity; illumination 06: 00 to 18: 00.

pharmacokinetic model

Prior to administration, the test compounds were formulated by dissolving in sterile 0.9% saline to make a 5mg/ml preparation. A single dose of 2mg/kg of test compound was administered orally to dogs. Blood samples (4.5ml) were taken from the head vein at 0.25, 0.5, 0.75, 1, 2, 3, 4 and 6 hours after administration. Samples were collected in citrated Vacutainer tubes and kept on ice prior to centrifugation to sediment plasma. Plasma samples were derivatized with dinitrophenylhydrazine and analyzed by HPLC (Zorbax SB-C8 column) with methanol/500 mM sodium acetate adjusted to pH7 with phosphoric acid (60: 40, vol/vol). The plasma concentration of the test compound was recorded and used to calculate the pharmacokinetic parameters: release rate constant, Ke; total clearance, Clt; volume of distribution, V_D(ii) a Time to maximum plasma concentration of test compound, Tmax; compound at maximum concentration or Tmax, Cmax; half life of plasma, t_0.5(ii) a And area under the curve, a.u.c; fraction of test compound absorbed, F.

Dog model for thrombosis in coronary animals

Surgical preparation and manipulation of dogs is described in Jackson et al, Circulation, 82, 930-. Using the pentobaryl ratioSodium (30mg/kg, i.v.) was anesthetized to a hybrid hound dog (6-7 months old, sex unlimited, Hazelton-LRE, Kalamazoo, Mich., USA), catheterized and vented to room air. Adjusting the volume and respiratory rate of the variant to maintain PO in the blood₂、PCO₂And a pH within the normal range. Subcutaneous probe electrodes were inserted to record lead II ECGs.

The left mid-lateral neck was dissected to isolate the left jugular vein and the common carotid artery. A pre-calibrated milliar puncture device (model MPC-500, milliar instruments, Houston, TX, usa) was inserted into the carotid blood vessel and Arterial Blood Pressure (ABP) was continuously monitored. In the experiment, a jugular vein was cannulated for blood sampling. In addition, the femoral veins of both hind legs were cannulated for administration of test compounds.

A left thoracotomy was performed in the 5 th intercostal space, and the heart was suspended from a pericardial stent. A 1-2cm segment of the left coronary artery branch (LCX) is isolated near the first major diagnostic ventricular branch. A3-4 mm long 26-gauge sharp needle wire cathode electrode (Teflon coated, 30-gauge silver coated copper wire) was inserted into the LCX and placed against the arterial vein (confirmed after the experiment was completed). The stimulation circuit is completed by placing an anode in a subcutaneous position. An adjustable plastic occluder is placed around the LCX over the electrode area. A pre-calibrated electromagnetic flow probe (Carolina Medical electronic, King, north Carolina, usa) was placed around the LCX near the cathode to measure coronary flow (CBF). After mechanical closure of 10-sLCX, the occluder was adjusted to suppress the hyperemic blood flow response by 40-50% of that observed. All hemodynamic and ECG measurements were recorded and analyzed with a data acquisition system (model M3000, Modular Instruments, Malvern, PA, usa).

Thrombosis and compound dosing regimens

And applying 100 muA Direct Current (DC) to the cathode to generate LCX intimal electric injury. The current was maintained for 60 minutes and then stopped whether the vessel was closed or not. Thrombosis occurred spontaneously until LCX if totally occluded (determined by zero CBF and S-T segment increase). Compound administration was started 1 hour after the aging closed the thrombus. The compounds of the invention were administered at doses of 0.5 and 1 mg/kg/hour with the initial 2 hour infusion, together with the injection of a thrombogenic agent (e.g., tissue plasma pro-enzyme activator, streptokinase, APSAC). Reperfusion was performed 3 hours after compound administration. The reocclusion of the coronary arteries after successful thrombolysis was defined as zero CBF and continued for ≥ 30 minutes.

Hematology and template bleeding time determination

Whole blood Cell count, hemoglobin and hematocrit values were measured with a hematology analyzer (Cell-Dyn 900, Sequoia-Turner, mountain View, Calif., USA) on 40. mu.l of a blood sample (1 part sodium citrate: 9 parts blood) plus sodium citrate (3.8%). A Simplate II bleeding time device (Organon Tekrika Durham, N.C., USA) was used to determine the bleeding time of the gingival template. Two horizontal incisions were made with the device on the left upper or lower gingiva of the dog. Each incision was 3mm wide and 2mm deep. After cutting, the bleeding time was calculated using a stopwatch. When the blood oozes out, the cotton pad is used for sucking. The time of the template bleeding is the time from the incision of the son to the stop of the bleeding. Bleeding times were taken before (0 min), at 60 min injection, at the end of the test compound administration (120 min) and at the end of the experiment.

All data were determined for significance using one-way analysis of variance (ANOVA) followed by Student-Neuman-kuelspot hoct test. ANOVA was repeatedly measured to determine significant differences between time points in the experiment. The data should be statistically different at least the P < 0.05 level. All values are mean ± SEM. All experiments were performed following the guidelines of the American society for Pathology. Further description of this approach is described in Jackson et al, J.Cardiovasc.Pharmacol., 21, 587-.

In the template bleeding time assay, the compounds of formula I of the present invention were also evaluated at 0.25, 0.50 and 1.0mg/kghr for the template bleeding time assay.

A positive result of any of the above tests confirms the utility of the compounds of the present invention.

Assays demonstrating inhibition of autoimmune disease

Detection 1

This was done as described in Holmdahl et al, immunology in clinical experiments (Clin. exp. Immunol.), 70, 373-378(1987), incorporated herein by reference. Approximately 4-30 female mice, 8-10 weeks old, were ovariectomized. The administration of the compounds of the invention was started within three weeks after castration in the experimental groups. One week after administration of the compound of formula I, immunization was performed with murine type II collagen. Rats are classified according to the clinical severity of arthritis, see Holmdahl et al, rheumatoid arthritis, 29, 106(1986), incorporated herein by reference. Sera were collected and analyzed for anti-type II collagen activity antibodies. At the end of the experiment, splenocytes were taken from the mice to determine T cell activity.

This activity can be demonstrated by determining a reduction in anti-collagen type II antibody titres using a conventional ELISA assay. Quantification of DNA synthesis by thymidine uptake allows assessment of reduced T cell reactivity to type II collagen administered to splenic T-cells by antigen-producing cells. Finally, the clinical severity of the disease is assessed daily by erythema first symptoms and swelling of one or more limbs. Clinical evaluation is related to histological examination.

Detection 2

4-30 young female Sprague-Dawley rats were fed animal feed and water ad libitum. The experimental group received the compound of formula I and all mice received generally the mouse cord, see Amason et al, Arch. neurol., 21, 103-108(1969), incorporated herein by reference. Mice were classified according to the symptoms of experimental immune encephalomyelitis. After about 3 to 7 weeks of administration of the compound of formula I, the mice are sacrificed and their spinal cords are removed and examined.

Activity is demonstrated by the ability of the compound to inhibit EAE.

Detection 3

5 to 50 mice (MRL/Ipr and NZB) were used. The evaluation parameters were: anti-DNA antibodies quantified by ELISA, as well as survival time and renal histology. The compounds of the invention were administered to mice and disease progression was assessed using the parameters described above.

Detection 4

5-15 women were selected for clinical study. Women were in general after amenorrhea, i.e. had amenorrhea for 6-12 months before the start of the test, had an autoimmune disease showing symptoms, but were otherwise healthy. Because of the idiosyncratic and subjective nature of these diseases, placebo-controlled groups were studied, i.e. women were divided into two groups, one of which received the compound of formula I as the active substance and the other of which received a placebo. The experimental group women take 50-200 mg of the medicine orally every day. The treatment lasts for 3-12 months. The number and extent of symptoms in both groups were kept accurately documented and the results compared at the end of the study. Not only were the results compared between the two groups of members, but the results for each patient were also compared to the reported symptoms for each patient prior to the study initiation.

The utility of the compounds of formula I can be demonstrated by the positive effect of at least one of the tests described above.

Detection of myocardial ischemia protection against reperfusion injury

Ten female and male rabbits were treated with 1mg of the compound of formula I. After 15 minutes, the rabbits were anesthetized and their coronary arteries closed for 30 minutes, followed by 4 hours of reperfusion.

The control group was treated with vehicle.

Blue dye was used to assess the area of risk and tetrazolium dye was used to assess the area of infarction. The reduced area of the infarct zone relative to the control group indicates that the compound of formula I is effective in inhibiting reperfusion-damaged myocardial ischemia.

Test methods to demonstrate increased testosterone

60 normal males aged 62-75 years were selected for evaluation, and the selection criteria were as follows:

the following are suitable:

1. in the normal male category of the Life Insurance Table (metropolian Life Instrument Table) (appendix 1), the body weight was 90% to 130% of the medium ideal body weight.

2. The testosterone in the serum of the census is lower than 500 ng/dl.

3. Serum prostate specific antigen less than or equal to 4 ng/ml.

4. The clinical prostate examination is normal, and the ultrasound general examination has no suspicious prostate knots.

5. There were no major medical cases over the last two years, such as angina pectoris, myocardial infarction or angioplasty, no history of visceral cancer or no prostate cancer at any time over five years.

6. In normal screening, including normal cardiopulmonary examination, there is no condition for peripheral vascular or venular disease, or other systemic disease.

7. Reported by the adjudication laboratory, the following must be within 10% of the normal upper or lower limits: CBC (total blood count), including hemoglobin, hematocrit, and total WBC (white blood cell count).

Unsuitable ones:

1. smoking to male.

2. There has been a history of thromboembolism or pulmonary embolism at any time in the past.

3. Men who drink more than 2 units of alcohol per day are mostly equivalent to 2 cups of wine and 2 bottles of beer.

4. The electrocardiogram general survey has obvious clinical abnormality.

The study was conducted in a parallel design with a control group compared to two groups of 10 and 40 mg/day doses of the compound of formula I for 14 weeks. Humans were randomly assigned to either the control or compound groups. Testosterone levels were measured every two weeks and were purchased from Diagnostic Products, Inc. No. 5700, 96 Seidel, los Angeles, 90045, Calif., USA, using the RIA, Coat-a-Count kit. A statistically significant increase in testosterone levels compared to the control group indicates that the compound of formula I is effective in increasing serum testosterone.

Claims (14)

1. A method of inhibiting a pathological condition selected from the group consisting of: alzheimer's disease, premenstrual syndrome, thrombomodulin deficiency, uterine fibrosis, excessive myeloperoxidase activity, hyperchromboemia, autoimmune diseases, reperfusion injury of myocardial ischemia and testosterone deficiency, which comprises administering to a mammal suffering from said pathological conditions an effective amount of a compound of formula I and its optical and geometric isomers; and non-toxic pharmacologically acceptable acid addition salts, N-oxides, esters and quaternary ammonium salts thereof,

wherein,

a is selected from CH₂And NR;

b, D and E are independently selected from CH and N;

y is:

(a) phenyl optionally substituted by 1-3 substituents independently selected from R⁴Substituted with the substituent(s);

(b) naphthyl optionally substituted by 1-3 independently selected R⁴Substituted with the substituent(s);

(c)C₃-C₈cycloalkyl optionally substituted by 1-2 substituents independently selected from R⁴Substituted with the substituent(s);

(d)C₃-C₈cycloalkenyl optionally substituted with 1-2 groups independently selected from R⁴Substituted with the substituent(s);

(e) a five-membered heterocyclic ring containing a substituent selected from the group consisting of-O-, -NR-²-and-S (O)_nUp to two heteroatoms of (A) optionally substituted by 1 to 3 substituents independently selected from R⁴Substituted with the substituent(s);

(f) six-membered heterocyclic rings containing radicals selected from-O-, -NR²-and-S (O)_nUp to two heteroatoms of (A) optionally substituted by 1 to 3 substituents independently selected from R⁴Substituted with the substituent(s);

(g) bicyclic ring system obtained by condensation of a five-or six-membered heterocyclic ring containing a substituent selected from the group consisting of-O-, -NR-, and the phenyl ring²-and-S (O)_nUp to two heteroatoms of (A) optionally substituted by 1 to 3 substituents independently selected from R⁴Substituted with the substituent(s); z¹Comprises the following steps:

(a)-(CH₂)_pW(CH₂)_Q-；

(b)-O(CH₂)_pCR⁵R⁶-；

(c)-O(CH₂)_pW(CH₂)_q；

(d)-OCHR²CHR³-; or

(e)-SCHR²CHR³-; g is:

(a)-NR⁷R⁸；

(b)

wherein n is 0, 1 or 2; m is 1, 2 or 3; z²is-NH-, -O-, -S-or-CH₂-; optionally fused to one or two phenyl rings on adjacent carbons, optionally substituted independently on carbon atoms with 1-3 substituents, and optionally independently on N atom with R⁴Substituted with suitable substituents of suitable chemical nature; or

(c) Bicyclic amines containing 5 to 12 carbon atoms, either bridged or fused, and optionally independently selected from R⁴1-3 substituents of (A); or

Z¹And G may be in combination:

w is:

(a)-CH₂-；

(b)-CH＝CH-；(c)-O-；(d)-NR²-；(e)-S(O)_n-；(f)(g)-CR²(OH)-；(h)-CONR²-；(i)-NR²CO-；(j)(ii) a Or (k) -C ≡ C-; r is hydrogen or C₁-C₆An alkyl group; r²And R³Independently are: (a) hydrogen; or (b) C₁-C₄An alkyl group; r⁴The method comprises the following steps: (a) hydrogen; (b) halogen; (c) c₁-C₆An alkyl group; (d) c₁-C₄An alkoxy group; (e) c₁-C₄An acyloxy group; (f) c₁-C₄An alkyl sulfide; (g) c₁-C₄An alkylsulfinyl group; (h) c₁-C₄An alkylsulfonyl group; (i) hydroxy (C)₁-C₄) An alkyl group; (j) aryl radical (C)₁-C₄) An alkyl group;

(k)-CO₂H；

(l)-CN；

(m)-CONHOR；

(n)-SO₂NHR；

(o)-NH₂；

(p)C₁-C₄an alkylamino group;

(q)C₁-C₄a dialkylamino group;

(r)-NHSO₂；

(s)-NO₂；

(t) an aryl group; or

(u)-OH。

R⁵And R⁶Independently is C₁-C₈Alkyl or together form C₃-C₁₀A carbocyclic ring;

R⁷and R⁸Independently are:

(a) a phenyl group;

(b) saturated or unsaturated C₃-C₁₀A carbocyclic ring of (a);

(c) c containing up to two hetero atoms selected from-O-, -N-and-S-₃-C₁₀A heterocycle;

(d)H；

(e)C₁-C₆an alkyl group; or

(f) And R⁵Or R⁶Forming a 3-to 8-membered nitrogen-containing ring;

r, whether linear or cyclic⁷And R⁸May be arbitrarily and independently selected from C₁-C₆Alkyl, halogen, alkoxy, hydroxy and carboxy;

R⁷and R⁸The ring formed may be optionally fused with a benzene ring;

e is 0, 1 or 2;

m is 1, 2 or 3;

n is 0, 1 or 2;

p is 0, 1, 2 or 3;

q is 0, 1, 2 or 3.

2. The use of a compound of formula I and its optical and geometric isomers, and the non-toxic pharmacologically acceptable acid addition salts, N-oxides, esters and quaternary ammonium salts thereof, in the manufacture of a medicament for the inhibition of a condition selected from the group consisting of: alzheimer's disease, premenstrual syndrome, thrombomodulin deficiency, uterine fibrosis, excessive myeloperoxidase activity, hyperchrombin, autoimmune diseases, reperfusion injury of myocardial ischemia and testosterone deficiency,

wherein,

a is selected from CH₂And NR;

b, D and E are independently selected from CH and N;

y is:

(a) phenyl optionally substituted by 1-3 substituents independently selected from R⁴Substituted with the substituent(s);

(b) naphthyl optionally substituted by 1-3 independently selected R⁴Substituted with the substituent(s);

(c)C₃-C₈cycloalkyl optionally substituted by 1-2 substituents independently selected from R⁴Substituted with the substituent(s);

(d)C₃-C₈cycloalkenyl optionally substituted with 1-2 groups independently selected from R⁴Substituted with the substituent(s);

(e) a five-membered heterocyclic ring containing a substituent selected from the group consisting of-O-, -NR-²-and-S (O)_nUp to two heteroatoms of (A) optionally substituted by 1 to 3 substituents independently selected from R⁴Substituted with the substituent(s);

(f) six-membered heterocyclic rings containing radicals selected from-O-, -NR²-and-S (O)_nUp to two heteroatoms of (A) optionally substituted by 1 to 3 substituents independently selected from R⁴Substituted with the substituent(s);

(g) bicyclic ring system obtained by condensation of a five-or six-membered heterocyclic ring containing a substituent selected from the group consisting of-O-, -NR-, and the phenyl ring²-and-S (O)_nUp to two heteroatoms of (A) optionally substituted by 1 to 3 substituents independently selected from R⁴Substituted with the substituent(s);

Z¹comprises the following steps:

(a)-(CH₂)_pW(CH₂)_q-；

(b)-O(CH₂)_pCR⁵R⁶-；

(c)-O(CH₂)_pW(CH₂)_q；

(d)-OCHR²CHR³-; or

(e)-SCHR²CHR³-

G is: (a) -NR⁷R⁸；(b)

Wherein n is 0, 1 or 2; m is 1, 2 or 3; z²is-NH-, -O-, -S-or-CH₂-; optionally fused to one or two phenyl rings on adjacent carbons, optionally substituted independently on carbon atoms with 1-3 substituents, and optionally independently on N atom with R⁴Substituted with suitable substituents of suitable chemical nature; or

(c) Bicyclic amines containing 5 to 12 carbon atoms, either bridged or fused, and optionally independently selected from R⁴1-3 substituents of (A); or

Z¹And G may be in combination:w is: (a) -CH₂-；(b)-CH＝CH-；(c)-O-；(d)-NR²-；(e)-S(O)_n-；(f)

(g)-CR²(OH)-；(h)-CONR²-；(i)-NR²CO-；(j)(ii) a Or

(k) -C ≡ C-; r is hydrogen or C₁-C₆An alkyl group; r²And R³Independently are: (a) hydrogen; or (b) C₁-C₄An alkyl group; r⁴The method comprises the following steps: (a) hydrogen; (b) halogen; (c) c₁-C₆An alkyl group; (d) c₁-C₄An alkoxy group; (e) c₁-C₄An acyloxy group; (f) c₁-C₄An alkyl sulfide; (g) c₁-C₄An alkylsulfinyl group; (h) c₁-C₄An alkylsulfonyl group; (i) hydroxy (C)₁-C₄) An alkyl group; (j) aryl radical (C)₁-C₄) An alkyl group; (k) -CO₂H；(l)-CN；(m)-CONHOR；(n)-SO₂NHR；(o)-NH₂；(p)C₁-C₄An alkylamino group; (q) C₁-C₄A dialkylamino group; (r) -NHSO₂；

(s)-NO₂；

(t) an aryl group; or

(u)-OH。

R⁵And R⁶Independently is C₁-C₈Alkyl or together form C₃-C₁₀A carbocyclic ring;

R⁷and R⁸Independently are:

(a) a phenyl group;

(b) saturated or unsaturated C₃-C₁₀A carbocyclic ring of (a);

(c) c containing up to two hetero atoms selected from-O-, -N-and-S-₃-C₁₀A heterocycle;

(d)H；

(e)C₁-C₆an alkyl group; or

(f) And R⁵Or R⁶Forming a 3-to 8-membered nitrogen-containing ring;

r, whether linear or cyclic⁷And R⁸May be arbitrarily and independently selected from C₁-C₆Alkyl, halogen, alkoxy, hydroxy and carboxy;

R⁷and R⁸The ring formed may be optionally fused with a benzene ring;

e is 0, 1 or 2;

m is 1, 2 or 3;

n is 0, 1 or 2;

p is 0, 1, 2 or 3;

q is 0, 1, 2 or 3.

3. The use according to claim 1, wherein the compound of formula I is of the formula:wherein G is

Or

4. The use according to claim 2, wherein the compound of formula I is selected from the group consisting of:

cis-6- (4-fluoro-phenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol;

(-) -cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol;

cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol;

cis-1- [6 '-pyrrolidinoethoxy-3' -pyridyl ] -2-phenyl-6-hydroxy-1, 2, 3, 4-tetrahydronaphthalene;

1- (4' -pyrrolidinoethoxyphenyl) -2- (4 "-fluorophenyl) -6-hydroxy-1, 2, 3, 4-tetrahydroisoquinoline;

cis-6- (4' -hydroxyphenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol; and

1- (4' -pyrrolidine ethoxy phenyl) -2-phenyl-6-hydroxy-1, 2, 3, 4-four hydrogen isoquinoline.

5. The use of claim 2, wherein said pathological condition is alzheimer's disease.

6. The use of claim 2, wherein said pathological condition is thrombomodulin deficiency.

7. The use of claim 2, wherein said pathological condition is uterine fibrosis.

8. The use of claim 2, wherein said pathological condition is excessive myeloperoxidase activity.

9. The use of claim 2, wherein said pathological condition is hyperchrombosis.

10. The use of claim 2, wherein said pathological condition is an autoimmune disease.

11. The use of claim 2, wherein said pathological condition is reperfusion injury of myocardial ischemia.

12. Use according to claim 2, wherein said pathological condition is testosterone deficiency.

13. The use of claim 2, wherein said pathological condition is premenstrual syndrome.

14. The use of claim 2, wherein said pathological condition is pre-amenorrhea syndrome.